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-   -   Genomic DNA QC using Bioanalyzer? (http://seqanswers.com/forums/showthread.php?t=31229)

d00b9p 06-19-2013 10:24 AM

Genomic DNA QC using Bioanalyzer?
 
This might sound like a daft question, but if I was to run 1 ul of genomic DNA on a Bioanalyzer HS chip would I be able to detect sample degradation? I realise that high molecular weight genomic DNA will be far too large to be detected, but would any smaller weight 'smear' show up on the Bioanalyzer trace?

We have some insect extractions with very little DNA (~100 ng) from a whole insect. We have found running out the DNA on a gel to be quite a poor way of seeing the DNA quality - needing at least 30 ng and using Ethidium staining. While we can sometimes see the faint high molecular weight band, it is near impossible to see if there is much 'smear'. And the main problem is that we have so little DNA in the first place.

I wondered if using the Bioanalyzer may help, since it uses so little of the precious sample.

chevrm 06-19-2013 10:31 AM

I'd consider a 12000 chip if you're willing to burn 1uL...it should be able to visualize anything over 0.5ng/uL

Susanne 06-21-2013 05:57 AM

The HS chip is quite sensitive with regards to genomic DNA and overloading. So I would recommend not to load more than 50 ng/Ál, better around 10-15 ng/Ál if you want to quantify. There are several problematic aspects to it though - if your sample runs into the upper marker, your quantification will be biased, and if you load too much, the quantification will also be wrong.
Genomic DNA cannot enter the channels and it's possible that the chip doesn't run properly (if the gDNA is not degraded).
The 0.5 ng/Ál quoted for the 12000 chip is referring to this concentration in a single peak. You will not see this concentration in a smear sample (more like 5 ng/Ál required).


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